Increasing the impact of vertebrate scientific collections through 3D imaging: The openVertebrate (oVert) Thematic Collections Network.

The impact of preserved museum specimens is transforming and increasing by three-dimensional (3D) imaging that creates high-fidelity online digital specimens. Through examples from the openVertebrate (oVert) Thematic Collections Network, we describe how we created a digitization community dedicated to the shared vision of making 3D data of specimens available and the impact of these data on a broad audience of scientists, students, teachers, artists, and more. High-fidelity digital 3D models allow people from multiple communities to simultaneously access and use scientific specimens. Based on our multiyear, multi-institution project, we identify significant technological and social hurdles that remain for fully realizing the potential impact of digital 3D specimens.

A highly contiguous genome assembly for the Yellow Warbler (Setophaga petechia).

The Yellow Warbler (Setophaga petechia) is a small songbird in the wood-warbler family (Parulidae) that exhibits phenotypic and ecological differences across a widespread distribution and is important to California's riparian habitat conservation. Here, we present a high-quality de novo genome assembly of a vouchered female Yellow Warbler from southern California. Using HiFi long-read and Omni-C proximity sequencing technologies, we generated a 1.22 Gb assembly including 687 scaffolds with a contig N50 of 6.80 Mb, scaffold N50 of 21.18 Mb, and a BUSCO completeness score of 96.0%. This highly contiguous genome assembly provides an essential resource for understanding the history of gene flow, divergence, and local adaptation in Yellow Warblers and can inform conservation management of this charismatic bird species.

Combining Species Delimitation, Species Trees, and Tests for Gene Flow Clarifies Complex Speciation in Scrub-Jays.

Complex speciation, involving rapid divergence and multiple bouts of post-divergence gene flow, can obfuscate phylogenetic relationships and species limits. In North America, cases of complex speciation are common, due at least in part to the cyclical Pleistocene glacial history of the continent. Scrub-Jays in the genus Aphelocoma provide a useful case study in complex speciation because their range throughout North America is structured by phylogeographic barriers with multiple cases of secondary contact between divergent lineages. Here, we show that a comprehensive approach to genomic reconstruction of evolutionary history, i.e., synthesizing results from species delimitation, species tree reconstruction, demographic model testing, and tests for gene flow, is capable of clarifying evolutionary history despite complex speciation. We find concordant evidence across all statistical approaches for the distinctiveness of an endemic southern Mexico lineage (A. w. sumichrasti), culminating in support for the species status of this lineage under any commonly applied species concept. We also find novel genomic evidence for the species status of a Texas endemic lineage A. w. texana, for which equivocal species delimitation results were clarified by demographic modeling and spatially explicit models of gene flow. Finally, we find that complex signatures of both ancient and modern gene flow between the non-sister California Scrub-Jay (Aphelocoma californica) and Woodhouse's Scrub-Jay (Aphelocoma woodhouseii) result in discordant gene trees throughout the species' genomes despite clear support for their overall isolation and species status. In sum, we find that a multi-faceted approach to genomic analysis can increase our understanding of complex speciation histories, even in well-studied groups. Given the emerging recognition that complex speciation is relatively commonplace, the comprehensive framework that we demonstrate for interrogation of species limits and evolutionary history using genomic data can provide a necessary roadmap for disentangling the impacts of gene flow and incomplete lineage sorting (ILS) to better understand the systematics of other groups with similarly complex evolutionary histories. [Gene flow; genomics; introgression; phylogenomics; population genetics; species delimitation; species trees; systematics.].

A highly contiguous reference genome for the Steller's jay (Cyanocitta stelleri).

The Steller's jay is a familiar bird of western forests from Alaska south to Nicaragua. Here, we report a draft reference assembly for the species generated from PacBio HiFi long-read and Omni-C chromatin-proximity sequencing data as part of the California Conservation Genomics Project (CCGP). Sequenced reads were assembled into 352 scaffolds totaling 1.16 Gb in length. Assembly metrics indicate a highly contiguous and complete assembly with a contig N50 of 7.8 Mb, scaffold N50 of 25.8 Mb, and BUSCO completeness score of 97.2%. Repetitive elements span 16.6% of the genome including nearly 90% of the W chromosome. Compared with high-quality assemblies from other members of the family Corvidae, the Steller's jay genome contains a larger proportion of repetitive elements than 4 crow species (Corvus), but a lower proportion of repetitive elements than the California scrub-jay (Aphelocoma californica). This reference genome will serve as an essential resource for future studies on speciation, local adaptation, phylogeography, and conservation genetics in this species of significant biological interest.

De novo assembly of a chromosome-level reference genome for the California Scrub-Jay, Aphelocoma californica.

We announce the assembly of the first de novo reference genome for the California Scrub-Jay (Aphelocoma californica). The genus Aphelocoma comprises four currently recognized species including many locally adapted populations across Mesoamerica and North America. Intensive study of Aphelocoma has revealed novel insights into the evolutionary mechanisms driving diversification in natural systems. Additional insights into the evolutionary history of this group will require continued development of high-quality, publicly available genomic resources. We extracted high molecular weight genomic DNA from a female California Scrub-Jay from northern California and generated PacBio HiFi long-read data and Omni-C chromatin conformation capture data. We used these data to generate a de novo partially phased diploid genome assembly, consisting of two pseudo-haplotypes, and scaffolded them using inferred physical proximity information from the Omni-C data. The more complete pseudo-haplotype assembly (arbitrarily designated "Haplotype 1") is 1.35 Gb in total length, highly contiguous (contig N50 = 11.53 Mb), and highly complete (BUSCO completeness score = 97%), with comparable scaffold sizes to chromosome-level avian reference genomes (scaffold N50 = 66.14 Mb). Our California Scrub-Jay assembly is highly syntenic with the New Caledonian Crow reference genome despite ~10 million years of divergence, highlighting the temporal stability of the avian genome. This high-quality reference genome represents a leap forward in publicly available genomic resources for Aphelocoma, and the family Corvidae more broadly. Future work using Aphelocoma as a model for understanding the evolutionary forces generating and maintaining biodiversity across phylogenetic scales can now benefit from a highly contiguous, in-group reference genome.

Historical specimens and the limits of subspecies phylogenomics in the New World quails (Odontophoridae).

As phylogenomics focuses on comprehensive taxon sampling at the species and population/subspecies levels, incorporating genomic data from historical specimens has become increasingly common. While historical samples can fill critical gaps in our understanding of the evolutionary history of diverse groups, they also introduce additional sources of phylogenomic uncertainty, making it difficult to discern novel evolutionary relationships from artifacts caused by sample quality issues. These problems highlight the need for improved strategies to disentangle artifactual patterns from true biological signal as historical specimens become more prevalent in phylogenomic datasets. Here, we tested the limits of historical specimen-driven phylogenomics to resolve subspecies-level relationships within a highly polytypic family, the New World quails (Odontophoridae), using thousands of ultraconserved elements (UCEs). We found that relationships at and above the species-level were well-resolved and highly supported across all analyses, with the exception of discordant relationships within the two most polytypic genera which included many historical specimens. We examined the causes of discordance and found that inferring phylogenies from subsets of taxa resolved the disagreements, suggesting that analyzing subclades can help remove artifactual causes of discordance in datasets that include historical samples. At the subspecies-level, we found well-resolved geographic structure within the two most polytypic genera, including the most polytypic species in this family, Northern Bobwhites (Colinus virginianus), demonstrating that variable sites within UCEs are capable of resolving phylogenetic structure below the species level. Our results highlight the importance of complete taxonomic sampling for resolving relationships among polytypic species, often through the inclusion of historical specimens, and we propose an integrative strategy for understanding and addressing the uncertainty that historical samples sometimes introduce to phylogenetic analyses.

Dense Geographic and Genomic Sampling Reveals Paraphyly and a Cryptic Lineage in a Classic Sibling Species Complex.

Incomplete or geographically biased sampling poses significant problems for research in phylogeography, population genetics, phylogenetics, and species delimitation. Despite the power of using genome-wide genetic markers in systematics and related fields, approaches such as the multispecies coalescent remain unable to easily account for unsampled lineages. The Empidonax difficilis/Empidonax occidentalis complex of small tyrannid flycatchers (Aves: Tyrannidae) is a classic example of widely distributed species with limited phenotypic geographic variation that was broken into two largely cryptic (or "sibling") lineages following extensive study. Though the group is well-characterized north of the US Mexico border, the evolutionary distinctiveness and phylogenetic relationships of southern populations remain obscure. In this article, we use dense genomic and geographic sampling across the majority of the range of the E. difficilis/E. occidentalis complex to assess whether current taxonomy and species limits reflect underlying evolutionary patterns, or whether they are an artifact of historically biased or incomplete sampling. We find that additional samples from Mexico render the widely recognized species-level lineage E. occidentalis paraphyletic, though it retains support in the best-fit species delimitation model from clustering analyses. We further identify a highly divergent unrecognized lineage in a previously unsampled portion of the group's range, which a cline analysis suggests is more reproductively isolated than the currently recognized species E. difficilis and E. occidentalis. Our phylogeny supports a southern origin of these taxa. Our results highlight the pervasive impacts of biased geographic sampling, even in well-studied vertebrate groups like birds, and illustrate what is a common problem when attempting to define species in the face of recent divergence and reticulate evolution.

The drivers of tropical speciation.

Since the recognition that allopatric speciation can be induced by large-scale reconfigurations of the landscape that isolate formerly continuous populations, such as the separation of continents by plate tectonics, the uplift of mountains or the formation of large rivers, landscape change has been viewed as a primary driver of biological diversification. This process is referred to in biogeography as vicariance. In the most species-rich region of the world, the Neotropics, the sundering of populations associated with the Andean uplift is ascribed this principal role in speciation. An alternative model posits that rather than being directly linked to landscape change, allopatric speciation is initiated to a greater extent by dispersal events, with the principal drivers of speciation being organism-specific abilities to persist and disperse in the landscape. Landscape change is not a necessity for speciation in this model. Here we show that spatial and temporal patterns of genetic differentiation in Neotropical birds are highly discordant across lineages and are not reconcilable with a model linking speciation solely to landscape change. Instead, the strongest predictors of speciation are the amount of time a lineage has persisted in the landscape and the ability of birds to move through the landscape matrix. These results, augmented by the observation that most species-level diversity originated after episodes of major Andean uplift in the Neogene period, suggest that dispersal and differentiation on a matrix previously shaped by large-scale landscape events was a major driver of avian speciation in lowland Neotropical rainforests.

Phylogenomic species delimitation in microendemic frogs of the Brazilian Atlantic Forest.

The advent of next-generation sequencing allows researchers to use large-scale datasets for species delimitation analyses, yet one can envision an inflection point where the added accuracy of including more loci does not offset the increased computational burden. One alternative to including all loci could be to prioritize the analysis of loci for which there is an expectation of high informativeness. Here, we explore the issue of species delimitation and locus selection with montane species from two anuran genera that have been isolated in sky islands across the southern Brazilian Atlantic Forest: Melanophryniscus (Bufonidae) and Brachycephalus (Brachycephalidae). To delimit species, we obtained genetic data using target enrichment of ultraconserved elements from 32 populations (13 for Melanophryniscus and 19 for Brachycephalus), and we were able to create datasets that included over 800 loci with no missing data. We ranked loci according to their number of parsimony-informative sites, and we performed species delimitation analyses using BPP with the most informative 10, 20, 40, 80, 160, 320, and 640 loci. We identified three types of phylogenetic node: nodes with either consistently high or low support regardless of the number of loci or their informativeness and nodes that were initially poorly supported where support became stronger as we included more data. When viewed across all sensitivity analyses, our results suggest that the current species richness in both genera is likely underestimated. In addition, our results show the effects of different sampling strategies on species delimitation using phylogenomic datasets.

Museum genomics reveals the speciation history of Dendrortyx wood-partridges in the Mesoamerican highlands.

Natural history collections are increasingly valued as genomic resources. Their specimens reflect the combined efforts of collectors and curators over hundreds of years. For many rare or endangered species, specimens are the only readily available source of DNA. We leveraged specimens from a historical collection to study the evolutionary history of wood-partridges in the genus Dendrortyx. The three Dendrortyx species are found in the highlands of central Mexico and Central America south to Costa Rica. One of these species is endangered, and in general, Dendrortyx are secretive and poorly represented in tissue collections. We extracted DNA from historical museum specimens and sequenced ultraconserved elements (UCEs) and mitochondrial DNA (mtDNA) to assess their phylogeny and divergence times. Phylogenies built from hundreds to thousands of nuclear markers were well resolved and largely congruent with an mtDNA phylogeny. The divergence times revealed an unusually old avian divergence across the Isthmus of Tehuantepec in the Pliocene around 3.6 million years ago. Combined with other recent studies, our results challenge the general pattern that highland bird divergences in Mesoamerica are relatively young and influenced by the Pleistocene glacial cycles compared to the older divergences of reptiles and plants, which are thought to overlap more with periods of mountain formation. We also found evidence for monophyletic genetic lineages in mountain ranges within the widespread D. macroura, which should be investigated further with integrative taxonomic methods. Our study demonstrates the power of museum genomics to provide insight into the evolutionary histories of groups where modern samples are lacking.

Genome-wide signals of drift and local adaptation during rapid lineage divergence in a songbird.

The formation of independent evolutionary lineages involves neutral and selective factors, and understanding their relative roles in population divergence is a fundamental goal of speciation research. Correlations between allele frequencies and environmental variability can reveal the role of selection, yet the relative contribution of drift can be difficult to establish. Recently diversified taxa like the Oregon junco (Aves, Passerellidae, Junco hyemalis oreganus) of western North America provide ideal scenarios to apply genetic-environment association analyses (GEA) while controlling for population structure. Analysis of genome-wide SNP loci revealed marked genetic structure consisting of differentiated populations in isolated, dry southern mountain ranges, and less divergent, recently expanded populations in humid northern latitudes. We used correlations between genomic and environmental variance to test for three specific modes of evolutionary divergence: (a) drift in geographic isolation, (b) differentiation along continuous selective gradients and (c) isolation-by-adaptation. We found evidence of strong drift in southern mountains, but also signals of local adaptation driven by temperature, precipitation, elevation and vegetation, especially when controlling for population history. We identified numerous variants under selection scattered across the genome, suggesting that local adaptation can promote rapid differentiation when acting over multiple independent loci.

Phylogenomic insights into the diversification of salamanders in the Isthmura bellii group across the Mexican highlands.

Mountain formation in Mexico has played an important role in the diversification of many Mexican taxa. The Trans-Mexican Volcanic Belt in particular has served as both a cradle of diversification and conduit for dispersal. We investigated the evolutionary history of the Isthmura bellii group of salamanders, a widespread amphibian across the Mexican highlands, using sequence capture of ultraconserved elements. Results suggest that the I. bellii group probably originated in southeastern Mexico in the late Miocene and later dispersed across the Trans-Mexican Volcanic Belt and into the Sierra Madre Occidental. Pre-Pleistocene uplift of the Trans-Volcanic Belt likely promoted early diversification by serving as a mesic land-bridge across central Mexico. These findings highlight the importance of the Trans-Volcanic Belt in generating Mexico's rich biodiversity.

Genomic data reveal ancient microendemism in forest scorpions across the California Floristic Province.

The California Floristic Province (CFP) in western North America is a globally significant biodiversity hotspot. Elucidating patterns of endemism and the historical drivers of this diversity has been an important challenge of comparative phylogeography for over two decades. We generated phylogenomic data using ddRADseq to examine genetic structure in Uroctonus forest scorpions, an ecologically restricted and dispersal-limited organism widely distributed across the CFP north to the Columbia River. We coupled our genetic data with species distribution models (SDMs) to determine climatically suitable areas for Uroctonus both now and during the Last Glacial Maximum. Based on our analyses, Uroctonus is composed of two major genetic groups that likely diverged over 2 million years ago. Each of these groups itself contains numerous genetic groups that reveal a pattern of vicariance and microendemism across the CFP. Migration rates among these populations are low. SDMs suggest forest scorpion habitat has remained relatively stable over the last 21 000 years, consistent with the genetic data. Our results suggest tectonic plate rafting, mountain uplift, river drainage formation and climate-induced habitat fragmentation have all likely played a role in the diversification of Uroctonus. The intricate pattern of genetic fragmentation revealed across a temporal continuum highlights the potential of low-dispersing species to shed light on small-scale patterns of biodiversity and the underlying processes that have generated this diversity in biodiversity hotspots.

Hidden histories of gene flow in highland birds revealed with genomic markers.

Genomic studies are revealing that divergence and speciation are marked by gene flow, but it is not clear whether gene flow has played a prominent role during the generation of biodiversity in species-rich regions of the world where vicariance is assumed to be the principal mode by which new species form. We revisit a well-studied organismal system in the Mexican Highlands, Aphelocoma jays, to test for gene flow among Mexican sierras. Prior results from mitochondrial DNA (mtDNA) largely conformed to the standard model of allopatric divergence, although there was also evidence for more obscure histories of gene flow in a small sample of nuclear markers. We tested for these 'hidden histories' using genomic markers known as ultraconserved elements (UCEs) in concert with phylogenies, clustering algorithms and newer introgression tests specifically designed to detect ancient gene flow (e.g. ABBA/BABA tests). Results based on 4303 UCE loci and 2500 informative SNPs are consistent with varying degrees of gene flow among highland areas. In some cases, gene flow has been extensive and recent (although perhaps not ongoing today), whereas in other cases there is only a trace signature of ancient gene flow among species that diverged as long as 5 million years ago. These results show how a species complex thought to be a model for vicariance can reveal a more reticulate history when a broader portion of the genome is queried. As more organisms are studied with genomic data, we predict that speciation-with-bouts-of-gene-flow will turn out to be a common mode of speciation.

Implementing and testing the multispecies coalescent model: A valuable paradigm for phylogenomics.

In recent articles published in Molecular Phylogenetics and Evolution, Mark Springer and John Gatesy (S&G) present numerous criticisms of recent implementations and testing of the multispecies coalescent (MSC) model in phylogenomics, popularly known as "species tree" methods. After pointing out errors in alignments and gene tree rooting in recent phylogenomic data sets, particularly in Song et al. (2012) on mammals and Xi et al. (2014) on plants, they suggest that these errors seriously compromise the conclusions of these studies. Additionally, S&G enumerate numerous perceived violated assumptions and deficiencies in the application of the MSC model in phylogenomics, such as its assumption of neutrality and in particular the use of transcriptomes, which are deemed inappropriate for the MSC because the constituent exons often subtend large regions of chromosomes within which recombination is substantial. We acknowledge these previously reported errors in recent phylogenomic data sets, but disapprove of S&G's excessively combative and taunting tone. We show that these errors, as well as two nucleotide sorting methods used in the analysis of Amborella, have little impact on the conclusions of those papers. Moreover, several concepts introduced by S&G and an appeal to "first principles" of phylogenetics in an attempt to discredit MSC models are invalid and reveal numerous misunderstandings of the MSC. Contrary to the claims of S&G we show that recent computer simulations used to test the robustness of MSC models are not circular and do not unfairly favor MSC models over concatenation. In fact, although both concatenation and MSC models clearly perform well in regions of tree space with long branches and little incomplete lineage sorting (ILS), simulations reveal the erratic behavior of concatenation when subjected to data subsampling and its tendency to produce spuriously confident yet conflicting results in regions of parameter space where MSC models still perform well. S&G's claims that MSC models explain little or none (0-15%) of the observed gene tree heterogeneity observed in a mammal data set and that MSC models assume ILS as the only source of gene tree variation are flawed. Overall many of their criticisms of MSC models are invalidated when concatenation is appropriately viewed as a special case of the MSC, which in turn is a special case of emerging network models in phylogenomics. We reiterate that there is enormous promise and value in recent implementations and tests of the MSC and look forward to its increased use and refinement in phylogenomics.

Ultraconserved elements are novel phylogenomic markers that resolve placental mammal phylogeny when combined with species-tree analysis.

Phylogenomics offers the potential to fully resolve the Tree of Life, but increasing genomic coverage also reveals conflicting evolutionary histories among genes, demanding new analytical strategies for elucidating a single history of life. Here, we outline a phylogenomic approach using a novel class of phylogenetic markers derived from ultraconserved elements and flanking DNA. Using species-tree analysis that accounts for discord among hundreds of independent loci, we show that this class of marker is useful for recovering deep-level phylogeny in placental mammals. In broad outline, our phylogeny agrees with recent phylogenomic studies of mammals, including several formerly controversial relationships. Our results also inform two outstanding questions in placental mammal phylogeny involving rapid speciation, where species-tree methods are particularly needed. Contrary to most phylogenomic studies, our study supports a first-diverging placental mammal lineage that includes elephants and tenrecs (Afrotheria). The level of conflict among gene histories is consistent with this basal divergence occurring in or near a phylogenetic "anomaly zone" where a failure to account for coalescent stochasticity will mislead phylogenetic inference. Addressing a long-standing phylogenetic mystery, we find some support from a high genomic coverage data set for a traditional placement of bats (Chiroptera) sister to a clade containing Perissodactyla, Cetartiodactyla, and Carnivora, and not nested within the latter clade, as has been suggested recently, although other results were conflicting. One of the most remarkable findings of our study is that ultraconserved elements and their flanking DNA are a rich source of phylogenetic information with strong potential for application across Amniotes.

Speciation in Western Scrub-Jays, Haldane's rule, and genetic clines in secondary contact.

BACKGROUND: Haldane's Rule, the tendency for the heterogametic sex to show reduced fertility in hybrid crosses, can obscure the signal of gene flow in mtDNA between species where females are heterogametic. Therefore, it is important when studying speciation and species limits in female-heterogametic species like birds to assess the signature of gene flow in the nuclear genome as well. We studied introgression of microsatellites and mtDNA across a secondary contact zone between coastal and interior lineages of Western Scrub-Jays (Aphelocoma californica) to test for a signature of Haldane's Rule: a narrower cline of introgression in mtDNA compared to nuclear markers. RESULTS: Our initial phylogeographic analysis revealed that there is only one major area of contact between coastal and interior lineages and identified five genetic clusters with strong spatial structuring: Pacific Slope, Interior US, Edwards Plateau (Texas), Northern Mexico, and Southern Mexico. Consistent with predictions from Haldane's Rule, mtDNA showed a narrower cline than nuclear markers across a transect through the hybrid zone. This result is not being driven by female-biased dispersal because neutral diffusion analysis, which included estimates of sex-specific dispersal rates, also showed less diffusion of mtDNA. Lineage-specific plumage traits were associated with nuclear genetic profiles for individuals in the hybrid zone, indicating that these differences are under genetic control. CONCLUSIONS: This study adds to a growing list of studies that support predictions of Haldane's Rule using cline analysis of multiple loci of differing inheritance modes, although alternate hypotheses like selection on different mtDNA types cannot be ruled out. That Haldane's Rule appears to be operating in this system suggests a measure of reproductive isolation between the Pacific Slope and interior lineages. Based on a variety of evidence from the phenotype, ecology, and genetics, we recommend elevating three lineages to species level: A. californica (Pacific Slope); A. woodhouseii (Interior US plus Edwards Plateau plus Northern Mexico); A. sumichrasti (Southern Mexico). The distinctive Edwards Plateau population in Texas, which was monophyletic in mtDNA except for one individual, should be studied in greater detail given habitat threat.

Applications of next-generation sequencing to phylogeography and phylogenetics.

This is a time of unprecedented transition in DNA sequencing technologies. Next-generation sequencing (NGS) clearly holds promise for fast and cost-effective generation of multilocus sequence data for phylogeography and phylogenetics. However, the focus on non-model organisms, in addition to uncertainty about which sample preparation methods and analyses are appropriate for different research questions and evolutionary timescales, have contributed to a lag in the application of NGS to these fields. Here, we outline some of the major obstacles specific to the application of NGS to phylogeography and phylogenetics, including the focus on non-model organisms, the necessity of obtaining orthologous loci in a cost-effective manner, and the predominate use of gene trees in these fields. We describe the most promising methods of sample preparation that address these challenges. Methods that reduce the genome by restriction digest and manual size selection are most appropriate for studies at the intraspecific level, whereas methods that target specific genomic regions (i.e., target enrichment or sequence capture) have wider applicability from the population level to deep-level phylogenomics. Additionally, we give an overview of how to analyze NGS data to arrive at data sets applicable to the standard toolkit of phylogeography and phylogenetics, including initial data processing to alignment and genotype calling (both SNPs and loci involving many SNPs). Even though whole-genome sequencing is likely to become affordable rather soon, because phylogeography and phylogenetics rely on analysis of hundreds of individuals in many cases, methods that reduce the genome to a subset of loci should remain more cost-effective for some time to come.

Ultraconserved elements anchor thousands of genetic markers spanning multiple evolutionary timescales.

Although massively parallel sequencing has facilitated large-scale DNA sequencing, comparisons among distantly related species rely upon small portions of the genome that are easily aligned. Methods are needed to efficiently obtain comparable DNA fragments prior to massively parallel sequencing, particularly for biologists working with non-model organisms. We introduce a new class of molecular marker, anchored by ultraconserved genomic elements (UCEs), that universally enable target enrichment and sequencing of thousands of orthologous loci across species separated by hundreds of millions of years of evolution. Our analyses here focus on use of UCE markers in Amniota because UCEs and phylogenetic relationships are well-known in some amniotes. We perform an in silico experiment to demonstrate that sequence flanking 2030 UCEs contains information sufficient to enable unambiguous recovery of the established primate phylogeny. We extend this experiment by performing an in vitro enrichment of 2386 UCE-anchored loci from nine, non-model avian species. We then use alignments of 854 of these loci to unambiguously recover the established evolutionary relationships within and among three ancient bird lineages. Because many organismal lineages have UCEs, this type of genetic marker and the analytical framework we outline can be applied across the tree of life, potentially reshaping our understanding of phylogeny at many taxonomic levels.

Temporal stability of the hybrid zone between Calocitta magpie-jays revealed through comparison of museum specimens and iNaturalist photos.

Hybrid zones are natural experiments for the study of avian evolution. Hybrid zones can be dynamic, moving as species adjust to new climates and habitats, with unknown implications for species and speciation. There are relatively few studies that have comparable modern and historic sampling to assess change in hybrid zone location and width over time, and those studies have generally found mixed results, with many hybrid zones showing change over time, but others showing stability. The white-throated magpie-jay (Calocitta formosa) and black-throated magpie-jay (Calocitta colliei) occur along the western coast of Mexico and Central America. The two species differ markedly in throat color and tail length, and prior observation suggests a narrow hybrid zone in southern Jalisco where individuals have mixed throat color. This study aims to assess the existence and temporal stability of this putative hybrid zone by comparing throat color between georeferenced historical museum specimens and modern photos from iNaturalist with precise locality information. Our results confirm the existence of a narrow hybrid zone in Jalisco, with modern throat scores gradually increasing from the parental ends of the cline toward the cline center in a sigmoidal curve characteristic of hybrid zones. Our temporal comparison suggests that the hybrid zone has not shifted its position between historical (pre-1973) and modern (post-2005) time periods-a surprising result given the grand scale of habitat change to the western Mexican lowlands during this time. An anomalous pocket of white-throated individuals in the northern range of the black-throated magpie-jay hints at the possibility of prehistorical long-distance introduction. Future genomic data will help disentangle the evolutionary history of these lineages and better characterize how secondary contact is affecting both the DNA and the phenotype of these species.